WO2006126066A2 - Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese - Google Patents

Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese Download PDF

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Publication number
WO2006126066A2
WO2006126066A2 PCT/IB2006/001353 IB2006001353W WO2006126066A2 WO 2006126066 A2 WO2006126066 A2 WO 2006126066A2 IB 2006001353 W IB2006001353 W IB 2006001353W WO 2006126066 A2 WO2006126066 A2 WO 2006126066A2
Authority
WO
WIPO (PCT)
Prior art keywords
erythropoietin
cells
nesp
dna
seq
Prior art date
Application number
PCT/IB2006/001353
Other languages
English (en)
Other versions
WO2006126066A3 (fr
Inventor
Patell Villoo Morawala
Original Assignee
Avestha Gengraine Technologies Pvt Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Avestha Gengraine Technologies Pvt Ltd. filed Critical Avestha Gengraine Technologies Pvt Ltd.
Priority to CA002609473A priority Critical patent/CA2609473A1/fr
Priority to AU2006250885A priority patent/AU2006250885A1/en
Priority to EP06744756A priority patent/EP1888630A2/fr
Priority to JP2008512940A priority patent/JP2009502117A/ja
Priority to AP2007004249A priority patent/AP2007004249A0/xx
Priority to BRPI0611405-9A priority patent/BRPI0611405A2/pt
Priority to US11/914,518 priority patent/US20090029907A1/en
Publication of WO2006126066A2 publication Critical patent/WO2006126066A2/fr
Publication of WO2006126066A3 publication Critical patent/WO2006126066A3/fr
Priority to IL187399A priority patent/IL187399A0/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention further relates to the optimized method for purification of the erythropoiesis stimulating protein.
  • novel biologically functional vital and circular plasmid DNA vectors incorporating DNA sequences of the invention and host organisms stably transformed or transfected with said vectors.
  • novel methods for the production of useful polypeptides comprising cultured growth of such transformed or transfected hosts under conditions facultative of large scale expression of the exogenous, vector-borne DNA-sequences and isolation of the desired polypeptides from the growth medium, cellular lysates or cellular membrane fractions.
  • Figure 6 Restriction digestion analysis of putative clones of AVCIPPCDNA3.1 D/V5- His/Nesp & AVCIPpcDNAS.lD/VS-His/Nesp-Opt.
  • Example 3 Sub-cloning of AVCIP-Nesp & AVCIP-Nesp-Opt cDNAs into the pcDNA3.1D/V5-His mammalian cell-specific expression vector
  • the de novo synthesized cDNA sequence original (AVCIP-Nesp) and codon optimized cDNA sequence (AVCIP-Nesp-Opt) were individually sub-cloned into the mammalian cell-specific expression vector pcDNA3.1D/V5-His to generate the transfection-ready constructs.
  • the details of the procedures used are given below:
  • Plasmid DNA was individually purified from the colonies obtained on L.B agar plates containing ampicillin and the presence of the desired cDNA insert was confirmed by restriction digestion analysis of the isolated plasmid DNA as shown in FIG No. 6.
  • AVCIPpcDNA3.1D/V5-His / Nesp & AVCIPpcDNA3.1D/V5-His / Nesp-Opt clones selected as a result of the restriction mapping analysis were further verified by automated DNA sequencing.
  • AVCIPpcDNAS.lD/VS-His/Nesp & AVCIPpcDNA3.1D/V5-His/Nesp-Opt clones showed 100% identity with the template sequence, as shown in FIG No. 8 & 9.
  • the maintenance and propagation of the cDNA construct encoding the novel erythropoiesis stimulating protein was carried out in a standard bacterial cell line such as Top 10 (Invitrogen).
  • Example 5 Transient / stable recombinant protein expression in CHO-Kl cells.
  • Total cell lysates were prepared from CHO Kl cell lines that were independently transfected with either pcDNA3.1/NESP (native) or pcDNA3.1/NESP(Opt Seq). The said cell lysates were prepared 48hrs after the transfection event and two different amounts of the total protein preparation (lOLIg and 20Hg) of the cell lysates were electrophoresed on a 12% SDS-PAGE prior to blotting on to a PVDF membrane. The PVDF membrane was then probed with 2I Jg/ml of rabbit anti-human erythropoietin antibody and the result obtained is shown in FIG No. 14.
  • Example 7 Purification of novel erythropoiesis stimulating protein
  • m-aminophenylboronic acid matrices are capable of forming temporary bonds with any molecule containing a 1,2-cis-diol group while Con A matrices bind specifically to mannosyl and glucosyl residues containing unmodified hydroxyl groups at the C3, C4 and C6 positions.
  • WGA Sepharose matrices are highly specific to N-acetyl glucosamine (NAG) or N-acetyl neuraminic acid (NANA or sialic acid) residues of the glycoprotein.
  • Reversed phase chromatography isoelectric focusing and two-dimensional gel electrophoresis will be employed to evaluate the purified product. Secondary structural analysis would be examined using far UV circular dichroism. Molecular mass and oligomeric status will be investigated using size exclusion and MALDI-TOF. The investigations will also focus on the stability of the protein in relation to pH and temperature. As NESP is a hyperglycosylated protein, glycosylation patterns of the purified protein would be documented using gas chromatography (GC) analysis.
  • GC gas chromatography

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne une méthode recombinante utilisée pour la production d'une forme hautement glycosylée (au total cinq glycosylations liées à N opposées à trois glycosylations liées à N dans l'EPO naturelle) d'érythropoïétine. Les sites ajoutés pour la glycosylation débouchent sur un nombre supérieur de chaînes de glucide, et un contenu d'acide sialique plus élevé que l'EPO humaine qui, à son tour, confère à la molécule recombinante une demi-vie plus longue. Cette invention a aussi trait à la construction de cassettes d'expression contenant des séquences d'acides nucléiques codant la forme hautement glycosylée d'érythropoïétine et l'expression stable dans les cellules hôtes. Cette invention a, également, pour objet la méthode optimisée destinée à la purification de la protéine de stimulation d'érythropoïése. L'EPO recombinante de ladite invention et les sels et les dérivés fonctionnels associés peuvent comprendre l'ingrédient actif de compositions pharmaceutiques pour augmenter l'hématocrite dans le traitement de l'anémie et dans la restauration du bien-être et de la qualité de vie d'un patient.
PCT/IB2006/001353 2005-05-24 2006-05-24 Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese WO2006126066A2 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
CA002609473A CA2609473A1 (fr) 2005-05-24 2006-05-24 Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese
AU2006250885A AU2006250885A1 (en) 2005-05-24 2006-05-24 A recombinant method for production of an erythropoiesis stimulating protein
EP06744756A EP1888630A2 (fr) 2005-05-24 2006-05-24 Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese
JP2008512940A JP2009502117A (ja) 2005-05-24 2006-05-24 赤血球新生刺激タンパク質生成のための組み換え法
AP2007004249A AP2007004249A0 (en) 2005-05-24 2006-05-24 A recombinant method for production of an erythropoiesis stimulating protein
BRPI0611405-9A BRPI0611405A2 (pt) 2005-05-24 2006-05-24 método recombinante para a produção de uma proteìna estimuladora da eritropoese
US11/914,518 US20090029907A1 (en) 2005-05-24 2006-05-24 Recombinant Method for Production of an Erythropoiesis Stimulating Protein
IL187399A IL187399A0 (en) 2005-05-24 2007-11-15 A recombinant method for production of an erythropoiesis stimulating protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN627CH2005 2005-05-24
IN627/CHE/2005 2005-05-24

Publications (2)

Publication Number Publication Date
WO2006126066A2 true WO2006126066A2 (fr) 2006-11-30
WO2006126066A3 WO2006126066A3 (fr) 2007-07-12

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2006/001353 WO2006126066A2 (fr) 2005-05-24 2006-05-24 Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese

Country Status (13)

Country Link
US (1) US20090029907A1 (fr)
EP (1) EP1888630A2 (fr)
JP (1) JP2009502117A (fr)
KR (1) KR20080026113A (fr)
CN (1) CN101228185A (fr)
AP (1) AP2007004249A0 (fr)
AU (1) AU2006250885A1 (fr)
BR (1) BRPI0611405A2 (fr)
CA (1) CA2609473A1 (fr)
IL (1) IL187399A0 (fr)
RU (1) RU2007147422A (fr)
WO (1) WO2006126066A2 (fr)
ZA (1) ZA200711011B (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011024024A1 (fr) * 2009-08-28 2011-03-03 Avesthagen Limited Procédé de récupération d'isoformes de la darbépoïétine alpha
EP2307442A2 (fr) * 2008-06-24 2011-04-13 Dr. Reddy's Laboratories Ltd. Purification de cytokines modifiées

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101443257B1 (ko) * 2011-10-18 2014-09-19 주식회사 종근당 낮은 등전점을 갖는 에리스로포이에틴 유사체의 정제방법
EP3194429A4 (fr) * 2014-09-18 2018-06-13 Askgene Pharma, Inc. Nouveaux agonistes du récepteur de l'érythropoïétine de félins
JP6906497B2 (ja) 2016-03-09 2021-07-21 Ｊｃｒファーマ株式会社 変異型ヒトエリスロポエチンの製造方法
WO2019084515A1 (fr) * 2017-10-26 2019-05-02 Essenlix Corporation Dispositifs et procédés pour la coloration de tissus et de cellules
WO2023040792A1 (fr) * 2021-09-14 2023-03-23 杰科（天津）生物医药有限公司 Procédé de préparation d'érythropoïétine

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2000024893A2 (fr) * 1998-10-23 2000-05-04 Amgen Inc. Methodes et compositions permettant de prevenir et de traiter l'anemie
WO2001081405A2 (fr) * 2000-04-21 2001-11-01 Amgen Inc. Methodes et compositions destinees a la prevention et au traitement de l'anemie

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Publication number Priority date Publication date Assignee Title
US5888774A (en) * 1994-12-19 1999-03-30 Cangene Corporation Recombinant DNA molecules and expression vectors for erythropoietin
US7304150B1 (en) * 1998-10-23 2007-12-04 Amgen Inc. Methods and compositions for the prevention and treatment of anemia
CA2424730A1 (fr) * 2000-10-02 2002-04-11 Arizeke Pharmaceuticals, Inc. Compositions et procedes pour le transport d'agents biologiquement actifs a travers les barrieres cellulaires

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2000024893A2 (fr) * 1998-10-23 2000-05-04 Amgen Inc. Methodes et compositions permettant de prevenir et de traiter l'anemie
WO2001081405A2 (fr) * 2000-04-21 2001-11-01 Amgen Inc. Methodes et compositions destinees a la prevention et au traitement de l'anemie

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EGRIE JOAN C ET AL: "Development and characterization of novel erythropoiesis stimulating protein (NESP)" NEPHROLOGY DIALYSIS TRANSPLANTATION, OXFORD UNIVERSITY PRESS, GB, vol. 16, no. Supplement 3, 2001, pages 3-13, XP001059695 ISSN: 0931-0509 *
ELLIOTT STEVE ET AL: "Enhancement of therapeutic protein in vivo activities through glycoengineering" NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 21, no. 4, April 2003 (2003-04), pages 414-421, XP002354911 ISSN: 1087-0156 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2307442A2 (fr) * 2008-06-24 2011-04-13 Dr. Reddy's Laboratories Ltd. Purification de cytokines modifiées
CN102076705A (zh) * 2008-06-24 2011-05-25 雷迪博士实验室有限公司 经修饰的细胞因子的纯化
EP2307442A4 (fr) * 2008-06-24 2012-05-30 Reddys Lab Ltd Dr Purification de cytokines modifiées
US8779110B2 (en) 2008-06-24 2014-07-15 Dr. Reddy's Laboratories Limited Purification of low isoelectric point isoforms of darbepoietin
WO2011024024A1 (fr) * 2009-08-28 2011-03-03 Avesthagen Limited Procédé de récupération d'isoformes de la darbépoïétine alpha

Also Published As

Publication number Publication date
ZA200711011B (en) 2008-11-26
KR20080026113A (ko) 2008-03-24
BRPI0611405A2 (pt) 2010-09-08
WO2006126066A3 (fr) 2007-07-12
EP1888630A2 (fr) 2008-02-20
AP2007004249A0 (en) 2007-12-31
AU2006250885A1 (en) 2006-11-30
JP2009502117A (ja) 2009-01-29
US20090029907A1 (en) 2009-01-29
CN101228185A (zh) 2008-07-23
IL187399A0 (en) 2008-02-09
CA2609473A1 (fr) 2006-11-30
RU2007147422A (ru) 2009-06-27

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